The present invention relates to methods and materials for detecting, clearing, or isolating parvovirus B19 and/or B19-like polypeptides from blood or other solutions containing it. The invention particularly provides polypeptides and recombinant bacteriophage expressing such polypeptides that are capable of binding to parvovirus B19 and/or B19-like polypeptides for the purpose of detecting, clearing, or isolating parvovirus B19 and/or B19-like viruses or polypeptides.
Parvoviruses form the parvoviridea family which are common agents of animal diseases. Parvovirus B19 is thus far the only strain identified to infect humans. The first strong link between parvovirus B19 infection and human disease was reported by Cossart et al. in England during screening of healthy blood donors for hepatitis B surface antigen. See, Cossart et al., Lancet, I: 72-73 (1975). xe2x80x9cB19xe2x80x9d refers to the designation of the sample from which this parvovirus was first isolated, and as the strain that is capable of infecting humans, it is often referred to as xe2x80x9chuman parvovirus B19xe2x80x9d.
Parvovirus B19 is a non-enveloped, single-stranded DNA virus with a diameter of 22 nm, consisting of only the genome and a few structural and non-structural proteins. The capsid proteins are arranged with icosahedral symmetry and enclose the genome of approximately 5500 base pairs. Two large open reading frames are in the viral genome: The left open reading frame codes for non-structural proteins (NS1 and NS2) involved in viral replication and packaging; the right open reading frame codes for the structural proteins forming the viral capsid, VP1(781 amino acids) and VP2 (554 amino acids). Both structural proteins are in the same reading frame and the entire sequence of VP2 is contained within VP1. VP2 is the major protein of the B19 capsid.
Parvovirus B19 is among the most resistant viruses known and has been identified as the causative agent of several diseases, including transient aplastic crisis (TAC) of hemolytic disease, the common childhood rash called xe2x80x9cfifth diseasexe2x80x9d, a polyarthralgia syndrome in normal adults that may be chronic and that resembles rheumatoid arthritis in its clinical features, and some forms of chronic anemia and/or neutrpenia. Pregnant women infected with this virus frequently suffer serious disabilities including spontaneous abortion and hydrops fetalis.
As a blood-borne virus, parvovirus B19 has become a concern for organizations dealing with whole blood or blood products intended, e.g., for use in transfusions. Therefore, it is important to develop sensitive methods for detection of the virus in infected blood and methods for clearing the virus from blood drawn from an infected subject.
Techniques employing Polymerase Chain Reaction (PCR) have become prevalent in recent years for detecting the presence of parvovirus B19 in biological samples. For example, Schwarz et al. utilized a pair of oligonucleotide primers spanning the PstI-fragment of the B19 virus genome to detect the B19 viral DNA in sera of individuals in the incubation period and acute phase of parvovirus B19 infection. See, Schwarz et al., Scand. J Infect. Dis., 24:691-696 (1992). See, also, Musiani et al., who utilized nested PCR to detect B19 infection in immunocompromised patients (J. Med. Virol., 40:157-160 (1993)), and also Torok et al., who employed PCR as a tool to diagnose prenatal intrauterine infection with parvovirus B19 (Clin. Infect. Dis., 14:149-155 (1992)).
Another approach taken to detect the presence of viral products in the infected individual is the use of in situ hybridization with detectable probes. For example, Morey et al. reported intracellular localization of parvovirus B19 nucleic acid by in situ hybridization with digoxiginin-labeled probes (Histochemical Journal, 25:421-429 (1993)). Later the same group employed a non-isotopic in situ hybridization technique in identifying parvovirus B19 infected cells using biotinylated probes (J. Clin. Pathol., 45: 673-678 (1992)). Although in situ hybridization is a rapid and specific means for localizing viral nucleic acid with a high degree of resolution, the sensitivity of this system is limited by the fact that hybridization occurs only at the surface of the section.
Further development of such assays has been hampered because parvovirus B19 cannot be isolated in conventional cell cultures and has only been propagated successfully in cultures of human bone marrow (Ozawa et al., Science, 233:883-886, (1986)), umbilical cord blood (Sosa et al., J Med. Virol., 36:125-130, (1992)), fetal liver (Yaegashi et al., J. Virol., 63:6,2422-2426, (1989)), and cultures from peripheral blood stimulated by erythropoietin (Schwarz et al., J. Virol., 66:1273-1276, (1992)). Another obstacle for development of such assays has been the possible existence of other parvoviruses and isotypes of parvovirus B19 that may also infect humans.
There is still a need, therefore, for sensitive and effective assays to detect the presence of B19 and/or B19-like viruses and subcomponents thereof, for ways to clear B19 and/or B19-like polypeptides from samples containing it (them), and for reagents that can bind B19 and/or B19-like polypeptides and which will be useful for detecting the presence of and/or clearing such viruses or polypeptides from samples, including blood.
In answer to the foregoing needs, a group of non-naturally occurring polypeptides has now been surprisingly discovered that bind specifically to parvovirus B19 and related polypeptides. Utilizing phage display technology, recombinant bacteriophage displaying polypeptides that recognize and bind to B19 capsid proteins have been identified and isolated. The phage products and isolated polypeptides have proved to be valuable reagents for effective detection and isolation of the B19 virus and B19-like polypeptides.
The present invention provides binding moieties for parvovirus B19 and/or B19-like viruses and polypeptide subcomponents of such viruses. Preferred binding moieties described herein are polypeptides and recombinant bacteriophage displaying such peptides which bind to parvovirus B19 and/or B19-like viruses, and most preferably to the virus""s capsid proteins VP1 and/or VP2.
In specific embodiments, the invention provides binding moieties for B19 and/or B19-like polypeptides as well as methods for detection and removal of human parvovirus B19 and/or B19-like polypeptides from samples (particularly human whole blood or blood products) containing it. In particular, preferred embodiments disclosed herein provide polypeptides that bind to parvovirus B19 capsid proteins VP1 or VP2 or combinations of such proteins and provide methods for binding and/or removing such capsid proteins from solutions containing them. Preferred features include recombinant bacteriophage expressing exogenous DNA encoding parvovirus B19 binding polypeptides.
A preferred binding moiety for human parvovirus B19 and/or B19-like polypeptides according to this invention will be a polypeptide having an amino acid sequence including a sequence selected from the group consisting of:
wherein X1 is Phe or Leu or is not present; X2 is Phe or Ser; X3 is Arg, Gln, Ser, His, Ala, Leu, or Gly; X4 is Phe, Tyr, Leu, or Trp; X5 is Trp or Phe; X6 is Tyr, Pro, or His; X7 is Gly, Asn, Ser, Phe, or Asp; X8 is His, Asp, Ser or Pro; X9 is Pro, Ala, Phe, His, or Asp or is not present;
wherein X10 is His, Ala, or Phe; X11 is His, Trp, or Ser; X12 is Phe or Leu; X13 is Phe, Pro, or His; X14 is Gly or His; X15 is Gly or Asn; X16 Pro, Leu, or Asp; X17 is His or Asp; and
wherein X18 is Phe or Leu; X19 is Trp, His, Gln or Pro; X20 is Leu or Ala; X21 is Trp or His; X22 is Pro or Trp; X23 is Ser, Ala, Pro or Gln; X24 is Ser, His, or Phe; X25 is Asp, Ser, Gln or Trp; and X26 is Phe, His, Ala or Asp.
Particularly preferred polypeptides of the invention include the following sequences:
Especially preferred embodiments include the polypeptides:
The present invention also provides binding moieties that are capable of binding human parvovirus B19 and/or B19-like viruses and dissociating from the virus under specific solution conditions. For example, preferred embodiments according to this invention bind to B19 at physiological pH and dissociate at low pH (e.g., pH 2).
Also included in the present invention are non-peptide and modified peptides that bind parvovirus B19 and/or parvovirus B19-like polypeptides. An example of these modifications is a constrained-loop peptide having paired cysteine residues that form disulfide bonds, modified at one cysteine residue by substitution of the cysteine with non-natural amino acid having a carboxylic acid side chain capable of condensing to form a stable thioester bridge. Such cyclic thioester analogues of synthetic peptides are described in PCT publication WO 97/46251, incorporated herein by reference. Other specifically contemplated modifications include N-terminal or C-terminal modifications of linkers such as poly-glycine segments and alterations to include functional groups, notably hydrazide (xe2x80x94NHxe2x80x94NH2) functionalities, to assist in immobilization of binding peptides according to this invention on solid supports.
The present invention also provides a method of detecting human parvovirus B19 and/or B19-like viruses in a solution suspected of containing it comprising the steps of contacting the solution with a binding moiety according to the invention and detecting whether binding of the peptide to the virus has occurred. The present invention also provides a method of removing human parvovirus from a solution containing it comprising the steps of immobilizing a B19 binding moiety on a chromatographic support, and contacting a solution containing human parvovirus B19 with the chromatographic material.
The present invention also provides a recombinant bacteriophage, i.e., bacteriophage transfected with exogenous DNA, that express one or more human parvovirus B19 binding peptides. Finally, the present invention provides a method for detecting human parvovirus B19 and/or B19-like polypeptides in a sample such as blood suspected of containing the virus, comprising the steps of contacting the blood with a bacteriophage expressing exogenous DNA encoding a human parvovirus B19 binding peptide, and detecting if binding has occurred between the peptide displayed on the bacteriophage and virus.
In the following sections, the term xe2x80x9crecombinantxe2x80x9d is used to describe non-naturally altered or manipulated nucleic acids, host cells infected with exogenous nucleic acids, or polypeptides expressed non-naturally, through manipulation of isolated DNA and transformation of host cells. Recombinant is a term that specifically encompasses DNA molecules which have been constructed in vitro using genetic engineering techniques, and use to the term xe2x80x9crecombinantxe2x80x9d as an adjective to describe a molecule, construct, vector, cell, polypeptide or polynucleotide specifically excludes naturally occurring such molecules, constructs, vectors, cells, polypeptides or polynucleotides.
As used herein, the term xe2x80x9cB19-like polypeptidexe2x80x9d refers to any subcomponent of parvovirus B19 or fragment of the whole B19 virus that is immunologically cross-reactive with parvovirus B19, including immunologically reactive fragments of the capsid and the tail. Capsid proteins VP1 and VP2, or combinations thereof, whether associated with the whole virus or isolated or synthetically prepared, are all specifically within the definition of the term xe2x80x9cB19-like polypeptidexe2x80x9d. The term also refers to other whole viruses and their subcomponents immunologically cross-reactive with B19, including all parvovirus B19 isotypes and any fragments of the isotypes, including but not limited to the isotypes that lead to clinical symptoms in humans.
The term xe2x80x9cbacteriophagexe2x80x9d is defined as a bacterial virus containing a DNA core and a protective shell built up by the aggregation of a number of different protein molecules.
The terms xe2x80x9cbacteriophagexe2x80x9d and xe2x80x9cphagexe2x80x9d are used herein interchangeably.
The term xe2x80x9cbinding moietyxe2x80x9d as used herein refers to any molecule, polypeptide, peptidomimetic or transformed cell (xe2x80x9ctransformantxe2x80x9d) capable of forming a binding complex with another molecule, polypeptide, peptidomimetic or tranformant. A xe2x80x9cB19 binding moietyxe2x80x9d is a binding moiety that forms a complex with parvovirus B19 or B19-like polypeptides. Specific examples of B19 binding moieties are the polypeptides mentioned above (SEQ ID NOs:1-23) and bacteriophage displaying any of such polypeptides. Also included within the definition of B19 binding moieties are polypeptides derived from a polypeptide having an amino acid sequence according to formula I, II or III, above, which have been modified for particular results (in addition to B19 or like polypeptide binding ability). Specific examples of modifications contemplated are COOHxe2x80x94or N-terminal amino acid substitutions or polypeptide chain elongations for the purpose of linking the binding moiety to a chromatographic support or other substrate, and substitutions of one or more cysteine residues that normally form disulfide links, for example with non-naturally occurring amino acid residues having reactive side chains, for the purpose of forming a more stable bond between those amino acid positions than the former disulfide bond. All such modified B19 binding moieties are also considered B19 binding moieties so long as they retain the ability to bind parvovirus B19 or B19-like polypeptides.